A full understanding of human oral health and disease requires a comprehensive analysis of the human oral microbiome and its interactions with the human host. A significant barrier to this goal is that 35% of the predominant human microbiota are uncultivable using standard cultivation methods. This proposal addresses the goals of RFA-DE-14-003: Innovative Approaches and Technologies for Examining the Uncultivated Bacteria of the Oral Microbiota through a multidisciplinary, team science approach, bringing together experts in microbiology, genomics, metabolomics, informatics, microfluidics and imaging. The specific aims have been constructed to achieve a systems-level integration of state-of-the-art technologies and innovative approaches. The proposal is hypothesis driven; it will provide specific knowledge on the nature of uncultivability for all uncultivated oral taxa identified in a subject population. This proposal will likely establish a new paradigm for cultivating previously uncultivable members of the oral microbiota by culturing first in consortia, rapidly reducing the complexity of the consortia, and bringing them into co-cultivation as binary pairs in commensal or syntrophic relationships. Clinical samples containing uncultivated bacteria will be incubated under novel growth conditions to enrich for reduced consortia containing uncultivables and helper species that supply essential nutrients. We will use bioinformatic and metabolic modeling approaches to determine the importance of auxotrophy and to identify novel growth factors. The proposal is the first to track genome evolution in studies to determine the genetic changes underlying bacterial domestication. Compounds that support the growth of previously uncultivable taxa will be identified and allow the formulation of novel culture media to support routine cultivation of a broader range of the oral microbiota. The identity of unknown nutrients will be determined by mass spectrometric methods. We will use novel micro-fluidic devices and spectral imaging technology to examine bacteria-bacteria and bacteria-host cell interactions of previously-uncultivable bacteria with other cells. Microfluidic systems will be used to quantify growth of uncultivable species in reduced consortia, in binary pairs, or when supplemented with a required metabolite or host factor. Periodontal disease studies using mock communities will provide an under-standing of the impact of previously-uncultivable bacteria on the commensal microbiota, the progression of disease, the host immune and inflammatory responses to pathogenic consortia, and the response of neutrophils and monocytes to previously-uncultivable bacteria alone or in pathogenic consortia. A web-accessible database on the prevalence, cultivation status, co-cultivation partners, auxotrophies, and domestication histories of previously-uncultivated taxa will be created for ready access to the scientific community. Isolates will be available for in vivo and in vitro studies of ecological interactions and interactions with the host. The high-throughput methods developed to cultivate currently- uncultivable oral bacteria will be generalizable to other body sites and to environmental microbiology.